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(Ultra)-structural morphology and biochemical parameters of liver samples from treated male and female rats, as well as fetuses and whole fetuses of treated females were investigated to provide information on molecular mechanisms responsible for toxic effects of the test item. 
Whole fetuses of treated dams as well as liver samples of treated dams and their fetuses of a preliminary oral teratogenicity study (Ciba-Geigy 924011) and a prenatal developmental toxicity study conducted according to OECD guideline 414 (Ciba-Geigy 924079) as well as liver samples of males treated within a one-generation reproduction toxicity study conducted according to OECD guideline 415 (Ciba-Geigy 894538) were investigated. The biochemical and (ultra)morphological investigations (Ciba-Geigy CB 92/02, Ciba-Geigy CB 92/14 and Ciba-Geigy CB 90/23 BY, respectively) identified the test item as a peroxisome proliferator in treated males, dams and the respective fetuses, the susceptibility to which is known to be a rodent specific phenomenon.
In addition, results of these studies provide a line of evidence that the increased peroxisomal metabolism generated substantial oxidative stress to tissues. The absence of fully developed or barely inducible defense mechanisms lead to the fetotoxic effects (skin discolorations, subcutaneous and skeletal muscular hemorrhages) of the test item seen in the prenatal developmental toxicity study.

Additional information

Within a prenatal developmental toxicity study conducted according to OECD guideline 414 (1981) doses of 0, 1, 30 and 150 mg/kg bw/d were administered to pregnant rats by gavage (see entry in IUCLID chapter 7.8.2; Ciba-Geigy 924079). Biochemical and morphological parameters in dam and fetal liver samples as well as whole fetuses obtained from control and high dose group upon necropsy at day 15, 18 and 21 were examined (Ciba-Geigy CB 92/14).

Major treatment related findings in dams consisted of hepatic peroxisome proliferation as evidenced by electron microscopy. This was confirmed biochemically by the induction of a number of diagnostic enzyme activities such as, lauric acid 11- and 12-hydroxylase activities, catalase activities and peroxisomal fatty acid beta-oxidation. No toxicologically relevant differences in hepatic glutathione status, liver content of malondialdehyde (product of lipid peroxidation) and activities of Se-dependent and Se-independent glutathione peroxidase were seen in treated dams.

Fetal livers from treated dams showed a striking peroxisome proliferation as early as day 15 of gestation as evidenced by electron microscopy. This finding was confirmed by induced activities of peroxisomal fatty acid beta-oxidation and lauric acid 11- and 12-hydroxylation.

In contrast to the respective dams low constitutive expression and poor or lacking inducibility of the protective fetal enzyme systems of catalase and glutathione peroxidases were seen. This presumably resulted in the decreased total hepatic glutathione content and the 2-fold increased liver malondialdehyde content at day 21 of gestation. Subcutaneous and skeletal muscular hemorrhages in fetuses from treated dams after 18 and 21 days of gestation were presumed to be the result of oxidative endothelial cell lesions. The lack of glycogen accumulation in fetuses up to day 21 of gestation was suspected to be due to oxidative stress as well.

 

These findings are supported by biochemical and ultrastructural investigations of livers of dams and fetuses as well as whole fetuses treated in a preliminary oral teratogenicity study (Ciba-Geigy 924011, IUCLID Chapter 7.8.2). Six females/dose/time point of satellite groups were treated from day 6 (p.c.) until necropsy on day 15, 18 and 21 with 0, 50 and 100 mg/kg bw/day. Livers of dams and fetuses as well as whole fetuses were collected and processed for biochemical and ultrastructural investigations (Ciba-Geigy CB 92/02).

Biochemical investigations of a series of parameters including protein content, catalase activity, peroxisomal beta-oxidation as well as oxidized and reduced glutathione level revealed changes which were strongly indicative of the action of the test article as peroxisome proliferator. This was confirmed by the ultrastructural findings which mainly included increased number and size of peroxisomes, some of which also showing matrical plate inclusion. In fetuses this was accompanied by the absence of fully developed defense mechanism as evidenced by the low catalase activity, diminished reduced glutathione status and the absence of glycogen stores. The concomitant fetal tissue lesions are therefore most probably attributable to oxidative damage as provoked by intensified peroxisomal metabolism.

 

In male rats peroxisome proliferation after treatment with the test substance was also confirmed. Within a one-generation reproduction toxicity study (OECD guideline 415, Ciba-Geigy 894538, IUCLID Chapter 7.8.1) male rats received single oral gavages at dose levels of 2, 50 and 100 mg/kg bw/d for 70 days prior to and throughout the mating period (21 days) until the start of parturition of dams. Male livers were collected and processed for biochemical and ultrastructural examinations. Biochemical investigation of a series of parameters included protein content, cytochrome P450 contents, enzymatic activities (e.g. morphine and bilirubin UGT, GST, monooxygenases) and peroxisomal beta-oxidation. Changes in the biochemical parameters (e.g. about 2 and 7-fold increased lauric acid 11- and 12-hydrocylation or about 8-fold increased peroxysomal fatty acid beta-oxidation) were strongly indicative for the action of the test article as peroxisome proliferator (Ciba-Geigy CB 90/23 BY). This was confirmed by the ultrastructural findings which mainly included changes in number and size of peroxisomes with matrical plate inclusions.

 

Thus, the biochemical and (ultra)morphological investigations identified the test item as a peroxisome proliferator in treated males, dams and the respective fetuses, the susceptibility to which is known to be a rodent specific phenomenon.

In addition, the results of this study provide a line of evidence in support of the hypothesis that substantial oxidative stress to fetal tissues as the consequence of strongly increased peroxisomal metabolism in the absence of fully developed or barely inducible defense mechanisms lead to the fetotoxic effects (skin discolorations, subcutaneous and skeletal muscular hemorrhages) of the test item seen in the prenatal developmental toxicity study.

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